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Fluorescence in situ Hybridization
Introduction to FISH Fluorescence in situ Hybridization (FISH) is a cytogenetic technique developed in the 1980's to analyze both DNA and mRNA. FISH uses fluorescent probes, which are sequences of DNA or mRNA, that bind to sequences with which they are complimentary. Then fluorescent microscopy can be used to examine where the probes bound to chromosomes, or to observe the amount of mRNA present. This technique is used in the field of medicine, genetic counsleing, karyotyping, tumor gene expression analysis, and the identification of species. This technique was also very important in the completion of the Human Genome Project. The Process (DNA) # A probe is constructed with flourophores using nick translation or by completing PCR with tagged nucleotides. The probe must be large enough to bind specifically to the hybridization point of interest, but not so large as to impede the binding. # Then a cell culture of prophase and metaphase cells are created using small DNA fragments to arrest mitosis. # Then the probe is applied and hybridization is allowed to occur for at least 12 hours. # Finally the substrate is washed to remove unbound and partially bound probes. # The presence of the probe bound to a complimentary piece of DNA is observed using fluorescent microscopy. # If the signal is weak, antibodies can be used to target the dye, and increase the fluorsesent signal. The Process (RNA) # A probe is constructed using at least 20 oglionucleotide pairs. # The probe is applied to a tissue sample and allowed to bind to the target RNA. # A multiplex assay is then used to increase the signal, along with amplification from further hybridization. # The tissue is then observed under a flourescent microscope to observe gene expression from the presence of target RNA. Analysis of Results FISH provides a picture of chromosomes or a tissue sample with glowing "marked" points. These points are where the probe has bound to complimentary sequences. This can show the researcher abnormalities such as increased gene expression, chromosomal rearrangement/deletion/trisomy, and in identifying chromosomes. FISH is a very general technique that has many other applications with different probes and in different substrates since "in situ" refers to marking DNA/mRNA within a cell. Specific Uses of FISH FISH's high sensitivity, specificity, and the speed at which the assays can be conducted have made it a huge discovery in genetic testing. It has become a very important resource for cytogenetic research and diagnosis. Some of the tests and research it can be used in are: # research and diagnosis of hematological tumors and solid tumors # detect genetic abnormalities such as fusions, aneuploidy, and deletion of chromosomal regions # diagnosis of genetic disorders and suggesting a prognosis # gene mapping experiments # identification of oncogenes # karyotyping # screening of whole genome for genetic aberration References Bishop, R. (2010). Applications Of Fluorescence In Situ Hybridization (FISH) In Detecting Genetic Aberrations Of Medical Significance. Bioscience Horizons, 3(1), 85-95 Fluorescence in situ hybridization. (2014, September 24). Retrieved September 29, 2014. Multiplex (assay). (2014, January 1). Retrieved September 29, 2014.